Larvicidal and repellent potential of Ageratum houstonianum against Culex pipiens

Mosquitoes are unquestionably the most medic arthropod vectors of disease. Culex pipiens, usually defined as a common house mosquito, is a well-known carrier of several virus diseases. Crude ethanol extracts of different organs of Agratum houstonianum are tested with Culex pipiens Linnaeus (Diptera: Culicidae) to determine their larvicidal, antifeedant, and repellency effects. Alongside biochemical analysis, the activity of the AChE, ATPase, CarE, and CYP-450 is detected in the total hemolymph of the C. pipiens larvae to examine the enzymatic action on the way to explain their neurotoxic effect and mode of action. Through HPLC and GC–MS analysis of the phytochemical profile of A. houstonianum aerial parts is identified. The larvicidal activity of aerial parts; flower (AF), leaf (AL), and stem (AS) of A. houstonianum extracts are evaluated against the 3rd instar larvae of C. pipiens at 24-, 48- and 72-post-treatment. A. houstonianium AF, AL, and AS extracts influenced the mortality of larvae with LC50 values 259.79, 266.85, and 306.86 ppm, respectively after 24 h of application. The potency of AF and AL extracts was 1.69- and 1.25-folds than that of AS extract, respectively. A high repellency percentage was obtained by AF extract 89.10% at a dose of 3.60 mg/cm2. A. houstonianium AF prevailed inhibition on acetylcholinesterase and decrease in carboxylesterase activity. Moreover, a significant increase in the ATPase levels and a decrease in cytochrome P-450 monooxegenase activity (− 36.60%) are detected. HPLC analysis prevailed chlorogenic and rosmarinic acid as the major phenolic acids in AL and AF, respectively. GC–MS analysis of A. houstonianum results in the identification of phytol as the major makeup. Precocene I and II were detected in AF. Linoleic, linolenic, and oleic acid were detected in comparable amounts in the studied organs. Overall, results suggest that the A. houstonianum flower extract (AF) exhibits significant repellent, antifeedant, and larvicidal activities.

Mosquitoes considered vectors to a wide variety of serious human diseases. The Culex pipiens is widely distributed in Egypt causing nuisance to humans and transmits several viral diseases 1 . It is the vector of West Nile virus 2 , Rift Valley fever virus 3 , Wuchereria bancrofti 4 , yellow fever 5 , filariasis 6 and other major public health problems worldwide which cause a significant human and animal mortality and morbidity in addition to sever economic losses. The mosquito control mainly based on the application of synthetic insecticides as larvicides or as adult repellents 7 . The chemical insecticides have adverse impacts on the health and environment beside to the development of resistance 8 . There is global interest in developing natural products as alternatives to conventional insecticides for mosquito control 9 . Many plant species have been screened for their repellent and insecticidal property 10 . Family Asteraceae contained many plant species which have been described for their medicinal and insecticidal purposes 11 . Ageratum houstonianum Mill. belonging to this family is a medicinal plant and possesses antimicrobial activity 10 . There are some previous reports on the insecticidal activities 12 of the different extracts of leaves of A. houstonianum as well as repellency against mosquitoes 13 . Furthermore, A. houstonianum has found to be a potent source of natural antioxidants 14 . Several classes of compounds were reported from A. houstonianum [15][16][17][18][19] . However, a literature survey has shown that there is no report on the phytochemicals of ethanolic extracts of different aerial parts (leaves, stems and flowers) of the Egyptian A. houstonianum which prompted authors to investigate the secondary metabolite profiles of the different organs under study. This study was planned to evaluate the larvicidal activity, repellant and antifeedant efficiency of ethanolic extracts of different GC/MS analysis of the lipoidal contents. It was concluded that the yield of lipoidal matter of leaves, stems, and flowers were (3.3%, 1.2% and 4.7%), respectively. The percentage of the unsaponifiable matter (USM) were (58.80%, 55.20% and 58.10%) and FAME were (38.20%, 33.70% and 40.40%) in the extracts of leaves, stems, and flowers, respectively. GC/MS analysis leads to identification of 30, 26 and 31components representing (99.27%, 99.33% and 97.50%) of the n-hexane extract yield of leaves, stems and flowers respectively ( Table 2, Fig. S2). It was observed that: unsaponifiable matter was composed of hydrocarbons, alcohols, ketones, aldehydes, esters, acids, phenols, sterols, chromenes, quinones, lactones and epoxides. The hydrocarbons represented (19.06%, 10.77 and 15.24%) of the USM of leaves, stems, and flowers respectively. The main of which was 5-Octadecene (3.07%) in leaves, 3-Eicosene (2.78%) in stem and in flowers was Tridecane, 5-methyl (6.92%). Biochemical activity. Activity of the enzymes, AChE, ATPase, CarE and CYP-450 were detected in the total hemolymph of the C. pipiens larvae treated with LC 50 of A. houstonianium flower, leaf and stem extracts were shown in Table 6. AChE activity was significantly inhabited in C. pipiens larvae, the obtained inhibition ratios of enzymatic activity ranging from − 57.86% (flower), − 40.979% (leaf) to − 15.95% (stem). It was noticed that both flower and leaf extracts have high inhibition efficacy against acetylcholinesterase than stem extract. All tested extracts led to decrease in the amount of CarE which more obvious with flower extract than other extracts. It was 43.12, 47.30 and 53.05 (ug Meb/min/mg protein) for flower, leaf, and stem, respectively, as compared with control 61.01 (ug Meb/min/mg protein).
Results given in Table 6 indicated that the tested extracts increase the amount of ATPase which was clearly detected in flower extract treatment compared with control. Amount of ATPase were 78.81, 69.16 and 63.93 (umoles Pi/min/mg protein) for extracts of flower, leaf, and stem, respectively, while it was 60.6 (umoles Pi/ min/mg protein) with control. A significant reduction in CYP-450 activity was obtained by treatment with all extracts whereas the flower extract showed the high reduction (− 36.606%) compared with leaf (− 22.14%) and stem (− 20.87%) extracts.

Discussion
Chlorogenic acid is one of the most abundant beneficial polyphenols in plants and is well known as nutritional antioxidant in plant -based foods. Apart from its dietary antioxidant activity, it has been proven to be an efficient defense molecule against a broad range of insect herbivores 20 . Increased efficiency of bio-insecticides is achieved by using chlorogenic acid as a synergistic bacterium. Chlorogenic acid has chemical defense against insects ascribed to its prooxidant effect by binding of the highly reactive chlorogenoquinone with nucleophilic-NH 2 and -SH groups in proteins and amino acids 21 . This reduces the bioavailability of amino acids consequently decreases digestibility of dietary proteins so, it considered as effective deterrent or anti-feedant 22 . www.nature.com/scientificreports/ High performance liquid chromatography (HPLC) and quantitative determination of phenolic contents of A. houstonianum showed that the ethanolic flowers extract was the richest extract in the flavonoid and total polyphenolic contents followed by the leaves then the stems, which interpreted the high potency of flowers extract than leaves followed by stem. This high potency was due to the synergism of its bioactive compounds which detected in high levels than in leaves and stems extracts. Where, the flowers extract exhibited high activity against C. pipiens larvae with approximately 2-folds than leaves and stems. The same results were detected for repellency and antifeedant effects against the C. pipiens adults. Where the repellency % obtained by flower extract was (89.1%) at a dose 3.6 mg/cm 2 indicating a good repellent property. Also, antifeedant activity and the maximum protection was obtained by flower extract with 90% of unfed females.
Regnault-Roger et al. 23 , showed that all phenolic compounds had toxicity to beetles, which paralyzed or dead at the bioassay test, by their cumulative toxic effect. Vanillin and caffeic and ferulic acids had a knockdown   www.nature.com/scientificreports/ effect, while rosmarinic acid, gallic acid, naringin and luteolin-7-glucoside had significant toxic and attractive effects. Rosmarinic acid was also detected at high concentration in the flowers. Rosmarinic acid is an insecticidal agent with high insecticidal activity at very low concentrations in 24 h against aphids. Also, it is known to reduce genotoxic effects induced by harmful chemicals so, it considered very safe to consumers 24 . The flavonoid rutin negatively affected the behavior, biology, and physiology of Spodoptera frugiperda and Helicoverpa zea by prolonging the larval development time, reducing the larval and pupal weight, and decreasing the pupal viability. The addition of different concentrations of rutin prolonged the life cycle of S. frugiperda; therefore, the use of rutin is indicated in future studies evaluating the control of S. frugiperda 25 . The flower extract showed higher total identified flavonoids than leaves and stems. Flavonoids and iso-flavonoids adversely affect insect growth, development, and behavior by influencing the steroid hormone systems. Some flavonoids are highly toxic to insect, while other act as feeding deterrents and repellency property 25 . The coumarin exhibited acute toxicity and deteriorated the growth of red palm weevil larvae 26 and showed antifeedant effects against Rhyzopertha dominica F. and Oryzaephilus surinamensis L. and demonstrated that the insect used the energy generated from ingested food to perform its physiological activities to fight the toxin (coumarin), therefore, affect the insect growth and development 27 . So, the polyphenols act in different ways and at different rate. Some components acted progressive toxicity while others had knockdown, repellent or anti-feedent effects.
Phytol was the major makeup in stem (52.10%), leaves (38.28%), and flowers 19.39%. Where, ketones represented by (4.65%, 4.69% and 5.12%) in the leaves, stems and flowers USM, respectively, the main of which was 2-pentadecanone, 6, 10, 14-trimethyl showing a yield of (2.05%) in the leaves and it is the only ketones present in the stems, while the main of which in flowers was 2-nonadecanone representing (2.34%). As well as aldehyde presented as (1.92% and 2.65%) in the USM of leaves and stems respectively, the main of which was palmitaldehyde diallyl acetal whose percentage was (1.83%) in stems and it is the only aldehyde detect in leaves. Furthermore, esters represented as (6.32%, 0.85% and 1.66%) in the leaves, stems and flowers USM, respectively. Acid and sterols were detected in comparable percent in the different organs under investigation. GC-MS analysis of the chloroform extract of Ageratum conyzoides whole plant prevailed 9,12-Octadecadienoic acid (12.48%), as major identified compound which comparable to our finding 28 .
Chromone presented by precocene II which was detected in leaves and stem as 22.08% and 13.26% respectively. While, in flower chromene I and II were detected. Chromone1 and 2 derivatives, detected in flower extract, are a well-known allelochemical and showed good insecticidal potency against M. separata 29 . Moreover, they have significant larvicidal activity against C. pipiens 30 . Also, these derivatives have antioxidant activity and MAOs inhibition activities 31 . These results agree with the present results, the flower extract exhibited higher insecticidal activity than stem and leaves against C. pipiens larvae.
Insecticidal effect of precocene II on the human body louse, Pediculus humanus was reported 32 . Essential oil of A. houstonianum Mill. aerial parts and its constituent compounds (precocene I and II) have potential for development into natural insecticides or repellents for control of insects in stored grains 33 . Precocene II inhibits juvenile hormone biosynthesis by cockroach corpora allata in vitro 34 . The precocenes (I and II), isolated from A. houstonianum, showed anti-juvenile hormonal effects on metamorphosis, ovarian development, and embryonic development also, exhibited larval mortality, the oviposition inhibition of ticks, Rhipicephalus microplus 35 . Fahmi et al. 36 , were investigate the influence of precocene II on the toxicological and biochemical parameters on the 4th instar larvae of S. littoralis. Overall, phytol can be considered further for developing effective and eco-friendly green insecticides against aphids 37 .
Whereas the ovicidal activity of A. houstonianum leaf extracts against the eggs of vector mosquitoes and to develop additional tools for the control of mosquito-borne diseases previously reported by Tennyson et al. 38 . The potential oviposition deterrent property of A. houstonianum crude leaf extracts detected in both laboratory and field studies designates the presence of phytocompounds that act as effective contact restraint 39 .
The insects have detoxification system to degrade toxic substances for the insect survival 40 . Metabolism of toxic substances involves two phases. The first phase is the cleavage of the substrate or addition of a polar group, while the second phase is the addition of sulfate, phosphate groups, sugar, or amino acid to the resulted products of 1st phase to increase hydrophilicity, consequently, facilitate excretion by the insect 41 . The most important enzymes responsible for the detoxification of toxins are CYP-450 for oxidative degradation and CarE for hydrolytic degradation that involved in 1st phase 42 . The detoxification capabilities of enzymes could be modified due to variations in gene expression 43 , consequently, variation of insect response to toxins 44 . The treatment of C. pipiens larvae with flowers, leaves and stem extracts inhibit the activity of CYP-450 and CarE activity with different levels due to variations in their constituents. The coumarin targets CYP-450 genes causing masking/silencing its expression that leads to high toxicity with low LD 50 values against red palm weevil 26 . These results agree with 45 who reported that the Piper betle extract reduced the level of CYP-450 in W strain of Ae. aegypti. Also, the sublethal dosage of A. conyzoides blocked the activity of CarE activity 46 . As well, the Sophora alopecuroides alkaloids are involved in the inhibition of CarE activity in Aedes albopictus 47 . In general, the esterases activities of the H. armigera larvae were significantly inhabited by flavonoid-treated diets 25 .
AChE has essential role as neurotransmitter in cholinergic synapses for insects 48 . Many insecticides inhibit of AChE action that causes accumulation of acetylcholine (ACh) at the synaptic cleft resulting in permanent neuro excitation/stimulation, paralysis, ataxia, and eventual death 49 . The obtained results showed that the flower and leaf extracts exhibited high inhibition effects against AChE than stem extract, that explained by Hussein et al. 30 , who proved that chromone 1 and 2 significantly inhibit the AChE activity in treated larvae of C. pipiens using molecular docking simulation. Many plant secondary metabolites decrease the levels of CarE and AChE activity of a wide range of insects 50 . The exposure of the A. aegypti larvae to the Sapindus emarginatus extract showed significant inhibition in the activities of AChE and CarE 51 , Similar reduction in AChE levels was observed by azadirachtin application against Nilaparvata lugens 52  www.nature.com/scientificreports/ ATPase plays a main role in intracellular functions and is a sensitive indicator of toxicity. It hydrolyzes adenosine triphosphate (ATP) to release the energy substantial for the active transport of Na + and K + across the cell membrane 53 . The metabolic detoxification mechanisms to toxins in insects consume high energy 54 . The elevated activity of the ATPase is a responsive action to the activation of detoxification mechanisms as a defense mechanism therefore, high energy demands 55 . Toxicity of botanical toxins to insects has been associated with the overexpression of genes involved with ATPase synthesis and energy demand 56 , this concept interpreted the enhancement of ATPase activity to reduce the damage caused by flower and leaves extracts, respectively, while the stem extract did not greatly stimulate ATPase with low expression.
Plant extracts have been studied extensively for their insecticidal effect 57 . Phytochemicals such as phenolic acid, flavonoids, chromene, phytol and monoterpenes are known for their mosquito repellent and insecticidal properties 57 . Ageratum houstonianum essential oil and extracts have been stated to have bioactive molecules 58 with repellency and adulticidal action against the adult mosquitoes 59 . There are various degrees of activity of Ageratum sp. extracts against insects due to variation of active ingredients with a wide variety of insecticidal properties 60 which agree with the results obtained in our investigation.
Many publications on the phytochemistry of Ageratum sp. from many disparate countries have been dealt with the various extracts with diversity in major and minor active constituents 61 . Petroleum ether extract of A. conyzoides showed significant larvicidal activity against the 4th larval instars, adult mortality and affected percentages of oviposition deterrence index of females of three mosquito vectors. Beside to, these extracts harmless to aquatic mosquito predator Toxorhynchites splendens even at the prominent dosage (1000 ppm) 46 . The A. conyzoides ethanolic extract has acaricidal potency against acaricides-susceptible and resistant ticks infesting buffaloes and cattle, moreover, adversely affected egg laying capacity 35 . Preparation of plant extract. One hundred grams of the powdered leaves, stems, and flowers of the plant were separately extracted with about 1000 ml of 70% ethanolic solution by using maceration till exhaustion then filtered. The collected extract was completely dried under vacuum using rotatory evaporator at 40 °C to yield a residue of about 30 g, 15 g and 25 g extracts for leaves, stems, and flowers, respectively. The extract was kept in tightly sealed containers to be used for the polyphenolic and biological study.

Materials and methods
Preparation of the n-hexane extracts. The powdered dried leaves, stems, and flowers (1000 g, 165 g and 150 g, respectively) of A. houstonianum were exhaustively extracted in a Soxhlet apparatus with n-hexane. The extracts were evaporated under reduced pressure at 40 °C to yield (35 g, 2 g and 7 g) greasy, dark green residue of leaves, stems, and flowers, respectively. The residues were stored in a desiccator for lipoidal matter investigation.
Preparation of the lipoidal matters. The lipoidal matters; unsaponifiable matter (USM) and fatty acid methyl esters (FAME) were prepared according to the method of Ichihara and Fukubayashi 62 , to identify the lipoidal constituents and to determine their percentages in the n-hexane extracts of leaves, stems, and flowers of A. houstonianum.
Spectrophotometric determination of total phenolic contents. The polyphenol content was determined using the Folin-Ciocalteu reagent method according to Mruthunjaya and Hukkeri 63 , with some modifications. The method involves the reduction of Folin Ciocalteau reagent (Sigma chemical, St.louis, Missouri, USA) by phenolic compounds, with a concomitant formation of a blue complex, and the absorbance was read at 765 nm using an UV-Vis spectrophotometer. The total polyphenolic content was expressed as gallic acid, using a standard calibration curve. Each experiment was repeated in triplicate and the readings were mean values. Same practice was repeated for the standard solution of gallic acid, and the calibration line was constructed. Based on the absorbance, the concentration of phenolics was interpreted (mg/ml) from the calibration line; then the contents of phenolics in extracts were articulated in the total phenolic contents as gallic acid correspondent (mg of GAE/g of sample).
Spectrophotometric determination of total flavonoid contents. Total flavonoid content was determined according to Atanassova et al. 64 , with some modifications. The absorbances of the solutions were measured at 510 nm against blank using a spectrophotometer. Similar procedure was returned for the standard solution of quercetin and the calibration graph was constructed. The content of flavonoids in each sample was articulate as quercetin, using a standard calibration curve as mg of QAE/g of sample).
HPLC analysis of the phenolic components. HPLC quantitative analysis of phenolic components was performed according to method presented by Mizzi et al. 65  Larvicidal bioassay. The 3rd arval instar of C. pipiens was treated with serial concentrations of A. houstonianum flower, leaf and stem extracts according to the previous standard protocol 67 with some modifications. Five concentrations of A. houstonianum flower, leaf and stem extracts were prepared in ethanol for stock solution, while serial concentrations (500, 400, 300, 200 and 100 ppm) were diluted using distilled water to prepare 100 ml of each concentration. Distilled water only was used for control. Twenty larvae were transferred to each treatment and control. Each treatment and control were replicated three times. Mortality was recorded after 24-, 48-and 72-h post-treatment.
Repellency and antifeedant bioassay. The standard cages (20 × 20 × 20 cm) were used to test the repellent activity of the extracts. Different amounts from each extract were dissolved in 2 ml (distilled water with a drop of Triton × 100) in 4 × 4 cm cups to obtain the different concentrations. The concentration was directly applied onto 5 × 6 cm of the ventral surface of pigeon after removing the abdomen's feathers. After 10 min of treatment, pigeons were placed for 3 h (from 6 to 9 PM) in cages containing the laboratory strain of starved C. pipiens females. Control tests were carried out using water. Each test was repeated three times to get a mean value of repellent activity 68 . Post treatment, the number of fed and unfed females was counted, and repellency was recorded statistically by using Abbott formula 69 .
where A: the percentage of unfed females in treatment. B: the percentage of unfed females in control.
Biochemical analysis. Enzyme preparation. The whole 3rd instar larvae of C. pipiens treated with LC 50 values were homogenized in distilled water (50 mg/1 ml). Homogenates were centrifuged at 8000 r.p.m. for 15 min at 5 °C in a refrigerated centrifuge. The deposits were discarded, and the supernatants were kept in a deep freezer (2 °C) till use as Amin 70 .
Acetylcholinesterase (AChE) activity assay. Acetylcholine bromide (AChBr) was used as substrate to detect the AChE activity according to the method described by Simpson et al. 71 . 200 µl enzyme solution were mixed with 0.5 ml AChBr (3 mm) and 0.5 ml 0.067 M phosphate buffer (pH 7). The mixture tubes were incubated for 30 min at 37 °C. Then 1 ml of alkaline hydroxylamine and 0.5 ml of HCl were added. The mixture tubes were mixed well and allowed to stand for 3 min. 0.5 ml of FeCl 3 solution was added to the mixture tube and shaken vigorously. The decrease in AChBr level resulted from the hydrolysis by AChE was read at 515 nm. www.nature.com/scientificreports/ ATPase activity assay. The total ATPase activity was estimated as described by Amaral et al. 72 . The main concept of this method is estimation the amount inorganic phosphate (Pi) resulted from ATP hydrolysis by ATPase. The enzyme was incubated at pH 7.5 and 37 °C, in 0.5 ml of a solution containing mixture of NaCl 150 mM, ATP.Na2-TRIS 5 mM and KCl 15 mM in histidine HCl-TRIS 30 mM. ATP was added to start the reaction. The mixture was incubated for 30 min at 37 °C, then 100 μl SDS (5%) was added to stop the reaction. The amount of formed Pi was measured by phosphorus kit. ATPase activity was expressed in µmoles of Pi released per minute per milligram protein.
Cytochrome P-450 monooxegenase (CYP-450) activity assay. P-nitroanisole O-demthylation was used to determine the CYP-450 activity according to Hansen and Hodgson 73 method with some modifications. The mixture solution containing 1.5 ml enzyme solution, 0.2 ml NADPH, 1 ml sodium phosphate buffer (0.1 M, pH 7.6), 50 µg glucose-6-phosphate dehydrogenase and 0.2 ml glucose-6-phosphate. p-nitroanisole in 10 µl of acetone was added to start the reaction and attain the final concentration of 0.8 mM. The final mixture was incubated at 37 °C for 30 min then 1 ml HCl (1 N) was added to terminate the incubation period. p-nitrophenol was extracted with 0.5 N NaOH and CHCl 3 . The absorbance of NaOH solution was estimated at 405 nm. An extinction coefficient of 14.28 mM/cm was used to calculate 4-nitrophenol concentration.
Carboxylesterase (CarE) activity assay. Carboxylesterase activity was determined as described by method of Simpson et al. 71 , and methyl n butyrate (MeB) used as substrate. The reaction solution containing 0.5 ml MeB (4 mM), 200 µl enzyme solution and 0.5 ml 0.067 M phosphate buffer (pH 7). The mixture tubes were incubated for 30 min at 37 °C. Then, 1 ml of alkaline hydroxylamine (equal volume of 3.5 M NaOH and 2 M hydroxylamine chloride) was added to the mixture tubes followed by 0.5 ml of HCl. The mixture tubes were mixed well and allowed to stand for 3 min. 0.5 ml of FeCl 3 solution was added to the mixture tube and shaken vigorously. The decrease in MeBr level resulted from the hydrolysis by carboxylesterases was read at 515 nm.
Statistical analysis. Lethal concentrations were determined at the 95% confidence level were recorded in probity regression line and LC 50 , and LC 90 , slope, standard error, and correlation coefficient; and for the goodness of fit (Chi square test) were calculated according to Finney 74 and correction for control mortality was conducted using Abbott's formula according to Abbott 69 . The biochemical results were analyzed by one-way analysis of variance (ANOVA) using CoStat system for Windows, Version 6.311 (CoHort software, Berkeley, CA 94701) https:// www. cohor tsoft ware. com/ costat. html. When the Anova statistics were significant (P < 0.01), means were compared by the Duncan's multiple range test 75 .

Conclusion
Overall, results suggest that the ethanolic ratios of enzymatic activity ranging extracts of flower, leaves, and stem of A. houstonianum exhibited a significant repellent, antifeedant and larvicidal activities with different levels, which may be attributed to chlorogenic, phytol, coumarin, rosmarinic acid, rutin, precocene I, and II compounds. All these bioactive molecules act in different ways with various rates and synergist each other to exhibit the toxicity action. Some components acted progressive toxicity while others had knockdown, repellent or anti-feedent effects. The flowers extract was rich with bioactive components which responsible for its high efficacy relative to leaves and stem extracts. The tested extracts inhibited the activity of AChE, CYP-450 and CarE with various levels, while the ATPase activity was enhanced. Different organs of A. houstonianum ethanol extracts could be used as bio-agents for mosquito control.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.